Preventing phytate salt deposition in polar  solvent systems

ABSTRACT

The present inventors have surprisingly discovered that phytic acid tenaciously precipitates with soluble metals in food or fuel ethanol-processing fluid, producing insoluble organometallic salt deposit or scale on the processing equipment that must be removed in order to facilitate further ethanol processing. The present invention relates to converting phytic acid salts or phytates to inorganic phosphates to improve metal solubility and reduce deposition within processing equipment.

This application is a Continuation application of U.S. Ser. No.11/873,630, filed Oct. 17, 2007, and which application is incorporatedherein by reference. A claim of priority, to the extent appropriate, ismade.

FIELD OF THE INVENTION

The present inventors have surprisingly discovered that the phytic acidtenaciously precipitates with soluble metals in food or fuelethanol-processing fluid, producing insoluble organometallic saltdeposit or scale on the processing equipment that must be removed inorder to facilitate further ethanol processing. The present inventionrelates to converting phytic acid salts or phytates to inorganicphosphates to improve metal solubility and reduce deposition withinprocessing equipment.

BACKGROUND

Fermentation of sugars and polysaccharides into alcohol is a rapidlydeveloping technology for producing liquid fuel, such as gasohol or E85,which are the most common examples in the United States and containvarying amounts of ethanol and gasoline. Billions of gallons of fuelethanol are produced every year through the fermentation of grains,plants and feedstock, primarily corn. Other types of feedstock such assugar care and cellulose are also increasing in importance.

Ethanol producers have found scale deposits on processing equipment atseveral stages of ethanol processing. These scale deposits are known toimpede heat transfer and flow, and interfere with the proper operationof mechanical devices used in ethanol processing. The deposits tend tobe most severe or tenacious on hot surfaces, and where the pH of theprocessing liquid is highest (about 4.5), but deposits may also form atlower pH values and on cooler surfaces. There remains a need for methodsand compositions for reducing this scale formation.

SUMMARY

The present inventors have unexpectedly discovered that phytic acidtenaciously precipitates with soluble metals in food or fuelethanol-processing fluid, producing insoluble organometallic saltdeposit or scale on the processing equipment that must be removed inorder to facilitate further ethanol processing. The present inventionrelates to converting phytic acid salts or phytates to inorganicphosphates to improve metal solubility and reduce deposition withinprocessing equipment.

In an embodiment, the present method can reduce insoluble depositformation in equipment that contacts food or fuel ethanol-processingfluids. The method can include: adding an agent to theethanol-processing fluids after fermentation; converting the insolublematerial to a soluble residue by action of the agent; and removing thesoluble residue from the equipment that contacts the ethanol-processingfluids. The method can also include identifying the insoluble depositfrom the ethanol-processing fluids.

In an embodiment, the present method includes adding an enzyme withphytase activity to the ethanol-processing fluids after fermentation;converting the phytate to orthophosphate by action of the enzyme; andremoving the soluble orthophosphate from the equipment that contacts theethanol-processing fluids. This embodiment can also include identifyingthe insoluble deposit from the ethanol-processing fluids as phytic acidor a salt of phytic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph depicting the difference in solubility of amagnesium phytate solution with and without treatment with phytase.

DETAILED DESCRIPTION Definitions

As used herein, the term “mash” refers to a mixture or slurry of milledgrain, process water and an enzyme such as alpha amylase, after themixture has been subjected to a high temperature pressure “cook” andintroduced into fermentation tank during ethanol processing.

“Cook water” refers to process water generated and/or used during cookprocess, where the starch content of milled grain is physically andchemically prepared for fermentation, typically by application of heatand by the action of enzymes such as amylase.

“Liquefaction” or “liquefy” means a process by which starch is convertedto shorter chain and less viscous dextrins. Generally, this processinvolves gelatinization of starch simultaneously with or followed by theaddition of enzymes such as amylase.

The term “liquefaction slurry” refers to combination of hot slurry, andthe slurries from primary and secondary liquefaction produced duringethanol processing. Hot slurry is formed when milled grain is firstmixed with process water and the formed slurry is treated with an enzymesuch as a-amylase and then heated to temperatures of up to 190° F. toreduce the viscosity of the slurry. The slurry is then pumped through apressurized jet cooker for flash condensation during primaryliquefaction. After flash condensation and cooling, the primaryliquefaction slurry is held at high temperature for one to two hours toprovide enough time for the amylase to fully break down the starch intoshort chain dextrins. During secondary liquefaction or“saccharification”, a second enzyme (such as glucoamylase) is added, andthe formed slurry is moved into fermentation tanks Liquefaction andsaccharification may take place successively or simultaneously.

“Fermentation” refers to a process by which the sugars in the slurry ormash from liquefaction/saccharification are converted into alcohol bythe action of yeast in the fermentation tanks or fermentors. The mash isallowed to ferment for 50-60 hours, resulting in a mixture that containsabout 15% ethanol as well as the solids from the grain and added yeast,i.e. the “fermentation slurry.” Once fermentation is complete, the mashor slurry is called “beer” and is moved into beer wells to be used forethanol distillation and recovery.

The term “whole stillage” refers to the mash or solids remaining afterethanol is removed from beer or beer mash using a stripper column. Theterm is used interchangeably with the term “thick stillage.” Wholestillage is typically 11% to 14% solids and contains all of the othernon-starch components of the grains that pass through the process (germ,protein, gluten, hull & fiber etc.).

“Thin stillage” refers to the liquid removed from the mash in ethanolproduction. Thin stillage is about 5% dry matter and about 95% water.Thin stillage can be reintroduced into the cooking and distillationprocesses to extract additional ethanol. Thin stillage that is recycledto the beginning of the dry-grind process is known as “backset” and isused to conserve water used in processing.

The term “beerstone” refers to a hard organometallic scale deposited onfermentation equipment and that is primarily calcium oxalate. Althoughbeerstone is a commonly formed deposit during ethanol processing, notall solid deposits formed are beerstone.

The term “phytase unit” refers to the amount of phytase enzyme that canliberate one micromole of ortho-phosphate from insoluble phytate in oneminute, assuming optimal conditions of temperature and pH.

Methods of the Invention

The commercial processing of ethanol produces aqueous slurries of plantgrains and fibers that release phytic acid. The present inventors haveunexpectedly discovered that the phytic acid tenaciously precipitateswith soluble metals in the processing fluid, producing insolubleorganometallic salt deposit or scale on the processing equipment thatmust be removed in order to facilitate further ethanol processing. Thepresent invention relates to converting insoluble phytic acid salts(i.e., phytate) to soluble inorganic phosphates and an organic compound(i.e., inositol), which can improve metal solubility and reducedeposition within processing equipment.

The present invention provides a method for reducing or even preventingthe formation of insoluble material, deposits, or scale on equipmentused in processing of food or fuel ethanol. According to the method, thedeposit or the material forming the deposit can be converted into asoluble material by the action of an agent capable of degrading orbreaking down the insoluble deposit or material that forms the deposit.The soluble material can then be easily removed from the equipment orprocessing system, by standard methods or conventional means known tothose of skill in the art. The method can include identifying content ofthe deposit.

In an embodiment, the method of the present invention includes reducingthe formation of insoluble deposits during ethanol processing. Duringcommercial food and fuel ethanol processing, the aqueous slurries ofplant grains and fibers produce acidic residues that interact withsoluble metals in ethanol-processing fluids to produce organometallicprecipitates. Many of these precipitates are insoluble solids thatdeposit as scale on processing equipment and interfere with downstreamprocessing of ethanol, by impairing heat transfer and causing productioninterruptions. In an aspect, the solid deposit formed in this manner isbeerstone, composed primarily of calcium oxalate. In another aspect, theaqueous slurries of plant grains and fibers form phosphate salts withdissolved metals present in ethanol processing fluid, such as salts ofmagnesium or calcium phosphate, for example. Various types of phosphatesalts can be formed during ethanol processing including, withoutlimitation, newberyite, bobierite, struvite (Mg salts), brushite,fluorapatite, hydroxyapatite (Ca salts), etc. In an aspect, thephosphate salt is a salt of a dissolved metal and phytic acid(C₆H₁₈O₂₄P₆; myoinositol hexakisphosphate, a phosphate ester ofinositol) that is released by the plant grains and fibers present in theaqueous slurries. In another aspect, the phosphate salt is magnesiumphytate. Phytate salts have been shown to form tenacious or insolubleprecipitates in the presence of polar protic solvents such as water andethanol, both of which are present in various concentrations duringethanol processing. Therefore, in an aspect, the method of the presentinvention provides for reducing or removing phytate that can deposit onethanol processing equipment.

In an embodiment, the method of the present invention includesidentifying the insoluble material formed during ethanol processing.Many different organometallic salts may be formed by the plant grainsand fibers present in ethanol processing fluids. The solubility productsof each salt may vary with processing conditions such as temperature andpH. Accordingly, the method can include chemically and/or geologicallyidentifying the deposit or materials susceptible to deposit, which canaid in reducing or removing the insoluble deposit from the processingequipment and processing fluids. The insoluble material may beidentified by standard methods known to those of skill in the art,including dry analysis methods such as x-ray fluorescence (XRF) oroxidation, followed by elemental analysis, for example, or wet analysismethods such as acid-base neutralization reactions, for example. In anaspect, the insoluble material is identified as a phosphate salt. Inanother aspect, the insoluble material is identified as a phytate salt,and in yet another aspect, the insoluble deposit is identified asmagnesium phytate.

A considerable amount of the phosphorus or phosphate content in plantgrains and fibers is in the form of phytic acid (as identified bystandard analysis methods such as high temperature sample oxidation toash), and commercial processing of these plant grains and fibers leadsto release of phytic acid. These phytic acid concentrations in liquids,such as ethanol-processing fluids, can be high enough to causeprecipitation of metal phytate salts, such as magnesium phytate, andsubsequent deposit formation, in ethanol processing equipment. Theformed phytates can impair heat transfer and cause productioninterruptions. A small amount of phytic acid is naturally broken downinto soluble byproducts (i.e., soluble phosphates) during fermentation,but a large quantity (i.e., approximately 30-35%) of the phosphorus orphosphate in the stillage and syrup remains as phytic acid or phytate.

Metal phytate salts are generally much less soluble than thecorresponding metal phosphates. For example, magnesium phytate is morethan an order of magnitude less soluble than magnesium phosphate, andtherefore, tends to precipitate out more readily than the more solublemagnesium phosphate. One way of causing phytate to precipitate is toheat a stable solution of magnesium phytate. Because phytate is lesssoluble at higher temperatures, a temperature is eventually reachedwhere precipitation occurs, and this temperature is lower than thetemperature at which magnesium phosphate would precipitate out. Thetemperature at which precipitation occurs is a function of pH andconcentration of magnesium and phosphate or phytate ions. Assumingsimilar pH conditions, a solution of magnesium phosphate must be heatedto about 40° C. more than a magnesium phytate solution, in order for thephosphate salt to precipitate out, even where the concentration ofmagnesium and phosphate ions were far greater than the concentration ofmagnesium and phytate ions, as shown in Table 1:

TABLE 1 Solubility of Magnesium with Phosphate and Phytate Solution pHTemp (° C.) Precipitate (+/−) 2100 ppm Mg²⁺ 5.2 40 − 8400 ppm PO₄ ³⁻ (asphosphate) 5.2 60 − 5.2 90 + 5.76 60 − 5.76 80 ++ 800 ppm Mg²⁺ 4.23 80 −3600 ppm PO₄ ³⁻ (as phytate) 4.52 60 − 4.52 80 + 4.97 40 − 4.97 60 ++5.36 21 − 5.36 40 ++ 5.6 21 +

Furthermore, the presence of ethanol also reduces the solubility ofmagnesium phytate. Table 2 indicates that precipitation of the phytatesalt occurred at much lower temperature in the presence of ethanol, evenat very similar pH. In the table, the water and ethanol columns indicatethe temperature at which a precipitate became visible in a solutioncontaining 800 ppm Mg²⁺ and 3600 ppm phytic acid.

TABLE 2 Solubility in Presence of Ethanol Temperature Temperature (° C.;in 100% (° C.; in 13% ethanol/87% pH water solution) water solution)4.35 40 4.52 80 4.53 30 4.6 21 4.97 60 5.36 40 5.6 21

In an embodiment, the present invention provides a method for reducingor removing insoluble material, such as phytate precipitates, by theaction of an agent added to ethanol processing fluids. In an aspect, theagent is an acidic compound that can break down organic phosphates andphosphonates into soluble inorganic phosphates in the presence of astrong oxidizer or oxidizing agent. For example, a persulfate candegrade the insoluble phytate through acid digestion. In another aspect,the agent is an acidic compound that, in combination with ultravioletlight, can break down organic phosphates and phosphonates into solubleinorganic phosphates. In an embodiment, the agent is an enzyme capableof digesting or degrading (e.g., hydrolyzing) organic phosphates orphosphonates into soluble inorganic phosphates and an organic compound.For example, the agent can be a phytase, which can hydrolyze phytate toinorganic phosphate and inositol.

Phytase is an enzyme known to be capable of breaking down the phyticacid found in plant material. It is currently used primarily in animalfeed applications, where it helps convert insoluble organic phosphatesinto soluble phosphorus that is more readily available to the animal'sdigestive system, and thereby also reduces environmental contaminationby insoluble phosphate salts such as phytates. In ethanol processing,the phytase has been used to increase the bioavailability of phosphorusfor the action of yeast in pre-saccharification and fermentation.Similarly, phytase has also been used in the liquefaction stage, orprior to fermentation to improve the activity of α-amylase.

Phytase is commercially available and can be derived from a variety ofsources. In an aspect, the phytase is obtained from plants ormicroorganisms, such as bacteria, or from fungi, such as yeast orfilamentous fungi, as disclosed in U.S. Patent Pub. No. 20050272137, forexample, and incorporated herein by reference. Plant phytases may bederived from wheat-bran, maize, soybean, or lily pollen. Bacterialphytases may be derived from various bacterial sources including,without limitation, Bacillus, Pseudomonas, or Escherichia, preferably B.subtilis or E. coli. In another aspect, the phytase is a yeast phytasederived from Saccharomyces or Schwanniomyces, preferably Saccharomycescerevisiae or Schwanniomyces occidentalis. In yet another aspect,phytases may be derived from filamentous fungi, including, but notlimited to, species from the genus Aspergillus, Thermomyces,Myceliophthora, Manascus, Penicillium, Peniophora, Agrocybe, and thelike.

Suitable commercially available phytases include, without limitation,those sold under the tradenames MAXALIQ™ ONE, by Genencor (Beloit,Wis.), RONOZYME® P5000 by Novozymes (Denmark), PHYTASE 5000L by DSM FoodSpecialties (France), NATUPHOS® 5000 by BASF (Germany), and PHYZYME™ XP10000 by Danisco Animal Nutrition (St. Louis, Mo.). Suitablecommercially available phytase enzyme may also be obtained fromsuppliers including, without limitation, Deerland Enzymes (Kennesaw,Ga.).

In an embodiment, phytase is added to ethanol processing equipmentand/or processing fluid at a time point and under conditions requiredfor the particular type of equipment or stage of ethanol processing. Inan aspect, phytase is added to fermentation fluid to reduce formation ofinsoluble deposits in downstream ethanol processing equipment. Inanother aspect, phytase is added to ethanol processing fluids downstreamof the fermentation process, such as, for example, beer, whole stillage,thin stillage, backset, centrate, or a mixture of these fluids. In anembodiment, phytase is added to thin stillage or backset. In an aspect,phytase is added to thin stillage or backset in line, i.e. the enzyme isintroduced directly into thin stillage or backset-containing equipmentduring ethanol processing. In another aspect, phytase is added to thinstillage or backset offline, i.e. the enzyme is added to thin stillageor backset contained in a separate vessel or tank. Phytase-treated thinstillage or backset can then be cycled back into processing lines fromthe vessel or tank.

In an embodiment, the present invention provides a method in which theagent or enzyme is introduced into the ethanol-processing fluid underoptimal conditions of temperature and pressure. Where the agent isphytase, the term “optimal conditions” refers to those conditions ofconcentration, temperature, residence time or reaction time, and pH thatallow sufficient reaction with soluble phytate, phytate suspension,phytate precipitate, or insoluble phytate scale that reduces the levelof the phytase deposit to an amount acceptable for operation of theethanol plant or process. In an embodiment, the conditions provide forcomplete hydrolysis of soluble phytate and phytate suspension.

In an aspect, the phytase is added to the ethanol processing fluid attemperatures of about 20° C. to about 80° C., for example, about 20° C.to about 77° C., about 40° C. to about 65° C., or about 30° C. to about55° C. (e.g., 52° C.). In an aspect, the phytase is added to the ethanolprocessing fluid at temperatures sufficient to allow the reactionbetween phytate and phytase to proceed to completion without degradingthe enzyme. In another aspect, the phytase is added to the ethanolprocessing fluid at pH of about 3 to about 9, for example, about 4.0 toabout 5.0, about 4.0 to about 5.5, or about 4.0 to about 5.3. In yetanother aspect, the phytase is added at a pH of 4.0, and the reaction isconducted at temperatures of about 40° C. to about 65° C., about 20° C.to about 77° C., or about 30° C. to about 55° C. (e.g., 52° C.).

In an aspect, the phytase is added to the ethanol processing fluids at aconcentration of about 100 ppm to about 500 ppm. In another aspect, thephytase is added at a concentration of 100 ppm, and in yet anotheraspect, the phytase is added at a concentration of 500 ppm. The phytasecan be added at concentrations expressed in phytase units. A unit ofactivity (U) is the amount of phytase that can release 1 μmol ofortho-phosphate per minute from excess phytic acid/phytate, at atemperature of 37° C. and a pH of about 5.5. Therefore, in an aspect,the phytase is added at a concentration of about 500 U/L to about 2500U/L. In another aspect, the phytase is added at a concentration of 100U/L, and in yet another aspect, the phytase is added at a concentrationof 2500 U/L.

In an embodiment, the phytase is added to ethanol processing fluids atlower concentrations, and the reaction is allowed to proceed over longerperiods of time. Extending the reaction time or residence time allowssmaller amounts of enzymes to be used, making ethanol processing moreeconomical. In an aspect, the phytase is added to the ethanol processingfluids for a residence time sufficient for complete reaction of thephytase with the insoluble phytate. In another aspect, the phytase isadded for a residence time of about 2 minutes to about 1200 minutes, forexample, about 3 minutes to about 200 minutes, or about 3 minutes toabout 40 minutes.

As shown in Table 3, low concentrations of phytase can releasesignificant amounts of ortho-phosphate, if reacted over longer periodsof time. For example, 5 ppm of phytase will release approximately 900ppm of ortho-phosphate when reacted over a 20 hour time period. Incontrast, 100 ppm of phytase releases 400-500 ppm of ortho-phosphate injust 10 minutes.

TABLE 3 Orthophosphate Formation at Low Doses of Phytase Amount ofReleased Ortho-Phosphate (ppm) Time (min) 5 ppm phytase 10 ppm phytase20 ppm phytase 20 73 92 109 60 143 232 387 140 260 436 732 260 377 636933 1200 894 1460 1713

Therefore, in an aspect, the phytase is added at a concentration ofabout 5 ppm to about 20 ppm. In another aspect, the phytase is added ata concentration of 5 ppm. In a further aspect, the phytase is added at aconcentration of 10 ppm, and in a yet further aspect, the phytase isadded at a concentration of 20 ppm. These concentrations may also beexpressed in phytase units such that, in an aspect, the phytase is addedat a concentration of about 25 U/L to about 100 U/L. In another aspect,the phytase is added at a concentration of 25 U/L. In a further aspect,the phytase is added at a concentration of 50 U/L and in a yet furtheraspect, the phytase is added at a concentration of 100 U/L.

In an embodiment, the present invention provides a method in which theagent added to the ethanol processing fluid converts soluble phytate,phytate suspension, phytate precipitate, or phytate scale into solubleorthophosphate. In an aspect, the agent is an enzyme such as phytase,which specifically hydrolyzes phytate. The enzyme can be used to convertphytate materials into soluble phosphates that are easily removed fromthe processing fluid, if necessary. Assuming identical conditions of pH(5.3) and identical concentrations of Mg²⁺ ions (1048 ppm) and phyticacid (4702 ppm), samples of processing fluid treated with phytaseremained clear (i.e., phosphates remain in solution withoutprecipitating out). However, samples of processing fluid not exposed tophytase showed progressively more precipitation of magnesium phytate asthe temperature is increased, as shown in Table 3 below.

TABLE 4 Enzyme Treatment Prevents Precipitation Enzyme Treated?Precipitate? (Y/N) Temp (° C.) (+/−) Y 20 − Y 40 − Y 80 − Y 100 − N 20 +N 40 ++ N 60 + N 80 +++

The following examples are provided to illustrate various aspects of theinvention, and should not be construed to limit the invention. A personof skill in the art will recognize that various modifications may bemade to the examples without departing from the scope of the presentinvention.

EXAMPLES Example 1 Conventional Analyses Indicate that Deposits in theBeer Column Contain Phosphorus, But Do Not Reveal That the Phosphorus isin Phytate

The content of various solid deposits formed during ethanol fermentationcan be determined using standard methods. It was previously thought thatthe solid deposits in the beer column were primarily beerstone (i.e.calcium oxalate). The unexpected results shown in the following examplesdemonstrate, however, that a large percentage of the solid depositsfound in the beer column are in the form of phosphates, i.e. P₂O₅.

Materials and Methods

To determine the content of the solid deposit in the beer column, X-rayfluorescence (XRF) analysis was used. A sample of solid deposit from thebeer column was collected and air-dried. A portion of the sample wasground to approximately 400 mesh using a steel swing mill, and theground sample was analyzed by XRF. Using standard XRF procedures, it waspossible to determine the presence of 31 major, minor and trace elementsto a relative precision/accuracy of approximately 5-10% for major andminor elements and approximately 10-15% for trace elements. A replicatesample was analyzed, along with a standard reference material (“SY3”, aCANMET standard rock or geological sample) to demonstrate analyticalreproducibility as well as analytical accuracy for a geologicalstandard.

Results

The content of the solid deposit in the beer column was as shown inTable 5. Major elements in the solid deposit include magnesium (as MgO)and phosphorus (as P₂O₅), while potassium and calcium (as theirrespective oxides K₂O and CaO) occur as minor elements. Trace amounts ofzinc were also detected. Major and minor elements were represented asweight percentages of the deposit, while trace elements were representedin ppm units.

TABLE 5 Deposit Analysis Element Content Magnesium (as MgO) 12.5 wt %Phosphorus (as P₂O₅) 35.7 wt % Potassium (as K₂O) 2.54 wt % Calcium (asCaO) 2.76 Zinc 18447 ppm

Conclusions

The results in Table 5 demonstrate that the majority of the soliddeposits are present as phosphate (rather than as the expected beerstoneor calcium oxalate). XRF analysis of solid deposits shows a largeconcentration of magnesium and phosphorus present in the oxide form, butdoes not distinguish between different chemical forms of phosphorus orphosphate salts, and does not specify if some of the magnesium wasactually present as a phosphate salt (i.e. magnesium phytate). Becausedifferent forms of phosphorus and phosphate salts have differentsolubilities and because the phytase has different activities ondifferent forms of phosphate, it is useful to distinguish between thevarious forms.

Example 2 The Deposits Include Substantial Amounts Phosphorus in Phytate

Because the activity of the phytase on the solid deposits is dependenton the type of phosphorus or phosphate salts present, wet analysis ofvarious deposit samples was used to determine the different forms ofphosphorus and phosphate salts present in the solid deposits of the beercolumn.

Materials and Methods

Deposit samples from ethanol plants were dissolved in a weighed amountof acid, the acid was neutralized and the solution was diluted to aknown volume. A portion of the neutralized sample was then subjected toa standard test for ortho-phosphate (PO₄ ³⁻), i.e. the Hach test. Totalphosphorus was determined by acid oxidation with persulfate, followed bythe reactive phosphorus test. Organically bound ortho phosphate was thendetermined by subtracting the acid-hydrolysable phosphorus content, andwas reported as PO₄ ³⁻ or ortho-phosphate.

A separate portion of the neutralized solution was treated with phytase(at a concentration of 500 ppm and 41° C. for 20-30 minutes). Underthese conditions, phytate present in the solution was converted toortho-phosphate. The amount of ortho-phosphate (PO₄ ³⁻) present afterphytase treatment represents the total phosphorus content of the deposit(total P as PO₄ ³⁻), i.e. original ortho phosphate and phytatephosphorus.

Results

As indicated in Table 6, deposit samples from an evaporator at oneplant, and samples from cook water lines and liquefaction pumps at asecond plant had between 60% and 75% of their phosphorus content asphytate, confirming that the deposit in the beer column includedprimarily magnesium phytate.

TABLE 6 Comparison of Deposit Analysis by XRF and Wet Methods Content byXRF Content by Wet Analysis Sample % MgO % P₂O₅ % PO₄ ³⁻ Total % P asPO₄ ³⁻ Plant 1 14.6 36.8 7.4 37.4 Evaporator (1) Plant 1 11.3 23.0 1.927.7 Evaporator (2) Plant 1 10.5 25.5 5.9 30.9 Evaporator (3) Plant 2Cook 14.0 35.5 2.4 36.0 Water Plant 2 Pump 16.7 35.2 9.1 38.0

Conclusions

The results in Table 6 demonstrate that the solid deposits were presentas phytate salt, i.e. magnesium phytate. This is an important discoverybecause the activity of the phytase added to various structures in anethanol plant depends on the presence of phosphate primarily in thephytate form. Solid deposits that exist primarily as phytate can bedissolved by the action of the phytase.

Example 3 Phytase Reduces the Phytate Concentration in Thin Stillage,Which is the Source of the Deposits Materials and Methods

The amount of phosphorus (present as ortho phosphate) in the thinstillage fraction collected from an ethanol plant will decrease oncephytase has been added. To determine the decrease in ortho-phosphateconcentration over time as a result of enzyme activity, thin stillagefractions were collected and treated with phytase at temperatures from43° C. to 62° C., at a pH of 4.0. The phytase concentration was either100 ppm or 500 ppm. After adding the enzyme, ortho-phosphateconcentration was measured at various time points (e.g., from 3 minutesto 20 minutes). Any phosphorus present as phytate (about 40-50% of thetotal phosphate) was converted to ortho phosphate (PO₄ ³⁻) by the actionof the phytase, with more phytate converted over increasing periods oftime. The concentration of ortho-phosphate in the thin stillage wastherefore a measure of enzyme activity.

Results

As demonstrated in Table 7, at an enzyme concentration of 500 ppm and atemperature of 43° C., the reaction was essentially complete in 5minutes, indicating that phytate was completely converted to orthophosphate. At an enzyme concentration of 100 ppm, and a temperature of62° C., the reaction continued for 20 minutes.

TABLE 7 Conversion of Phytate in Thin Stillage to Ortho PhosphateReleased Enzyme Phosphate PO₄ ³⁻ Conc. Temp Conc. Conc. Sample/Run (min)(ppm) (° C.) (ppm) (ppm) Distilled Water — — 0 — (zero) Std. 1 - 640 ppm— — 732 — PO₄ ³⁻ Std. 2 - 1280 ppm — — 1234 — PO₄ ³⁻ Run 1  5 min 50045.5 3313 1463 10 min 500 43.3 3385 1536 Std 2 1242 — Run 2  3 min 10063.3 1688 137  5 min 100 62.8 2146 595 10 min 100 62.2 2214 663 20 min100 61.0 2859 1309

Conclusions

The results shown in Table 7 indicate that substantially all of thephosphorus present in the phytate form was converted and released asortho-phosphate by the action of the phytase. About half of the totalphosphorus content of the thin stillage was in the form of phytate, andcomplete reaction of the enzyme with the phosphorus resulted in theconversion of phytate and release of ortho-phosphate. The amount orconcentration of released ortho-phosphate therefore provides a measureof the activity of the phytase.

Example 4 Increased Solubility on Conversion of Phytate toOrtho-phosphate

The solid deposits formed in various structures in an ethanol plantinclude primarily insoluble magnesium phosphate salts, includingmagnesium phytate. The following example illustrates that the solubilityof these salts is unexpectedly increased in the presence of phytase.

Materials and Methods

To determine the effect of phytase on increasing the solubility ofmagnesium phytate, two solutions of magnesium phytate were prepared bymixing magnesium salt with phytic acid in two separate test tubes. Thesolution in one test tube was treated with phytase, while the other wasleft untreated. The two solutions were maintained at the sametemperature and pH and had identical magnesium and total phosphoruscontent.

Results

The action of the phytase on magnesium phytate converted the insolublephytate salt into soluble ortho phosphate, which can then be readilyremoved from pumps, lines and evaporators in an ethanol plant. FIG. 1illustrates the differences in solubility of phytic acid solutions withand without the action of phytase.

The enzyme-treated solution in one test tube remained clear, indicatingthat all the phytate was converted to soluble ortho phosphate. Theuntreated solution showed precipitate formation, indicating the presenceof insoluble magnesium phytate. This demonstrated that the enzymecompletely converted the phytate to ortho-phosphate and greatlyincreased the solubility of the magnesium salt.

Conclusion

Phytase can prevent solid deposits of magnesium phosphate salts fromforming in various structures in an ethanol plant.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods, devices and material similar orequivalent to those described herein can be used in practice or testing,the methods, devices and materials are now described.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains and are incorporated herein by reference in theirentireties.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Those skilled in the art will readily recognize various modificationsand changes that may be made to the present invention without followingthe example embodiments and applications illustrated and describedherein, and without departing from the true spirit and scope of thepresent invention without following the example embodiments andapplications illustrated and described herein, and without departingfrom the true spirit and scope of the present invention, which is setforth in the following claims.

1-20. (canceled)
 21. A method of reducing formation of insoluble phyticacid salts in equipment that contacts food or fuel ethanol-processingfluids, the method comprising: fermenting plant grains to produce thinstillage, backset, or mixture thereof containing phytic acid salts;adding phytase to the thin stillage, backset, or mixture thereof andconverting the insoluble phytic acid salts to soluble products; therebyreducing the formation of deposits of insoluble phytic acid salts in theequipment that contacts the ethanol-processing fluids.
 22. The method ofclaim 21, wherein converting comprises partial conversion of phytate tophosphate and inositol.
 23. The method of claim 22, wherein convertingfurther comprises reacting the insoluble phytic acid salts in thepresence of polar protic solvents.
 24. The method of claim 23, whereinreacting the insoluble phytic acid salts in the presence of polar proticsolvents comprises reacting in the presence of ethanol and water. 25.The method of claim 21, further comprising adding the phytase tofermentation slurry, beer, whole stillage, centrate, or mixture thereof.26. The method of claim 21, wherein adding comprises introducing thephytase into the thin stillage, backset, or mixture thereof at atemperature from about 20° C. to about 80° C.
 27. The method of claim21, wherein adding comprises introducing the phytase into the thinstillage, backset, or mixture thereof at a pH of about 4.0.
 28. Themethod of claim 21, wherein adding comprises introducing the phytase fora residence time sufficient for complete reaction of the phytase withthe insoluble phytic acid salts.
 29. The method of claim 21, whereinadding comprises introducing the phytase for a residence time from about2 minutes to about 1200 minutes.
 30. The method of claim 21, furthercomprising analyzing an insoluble deposit in the equipment.
 31. Themethod of claim 30, wherein analyzing comprises detecting phytic acid orphytate.
 32. The method of claim 31, wherein analyzing comprisesdetecting magnesium phytate.
 33. A method of reducing formation ofinsoluble deposits of phytic acid or a salt of phytic acid in equipmentthat contacts food or fuel ethanol-processing fluids, the methodcomprising: adding an enzyme with phytase activity to thin stillage,backset, or mixture thereof after fermentation; converting the phytateto orthophosphate by action of the enzyme; and removing the solubleorthophosphate from the equipment.
 34. The method of claim 33, furthercomprising identifying the insoluble deposit in the ethanol-processingfluids as phytic acid or a salt of phytic acid.
 35. The method of claim33, further comprising adding the enzyme to fermentation slurry, beer,whole stillage, centrate, or mixture thereof.
 36. The method of claim33, wherein adding comprises introducing the enzyme into the thinstillage, backset, or mixture thereof at a temperature from about 20° C.to about 80° C.
 37. The method of claim 33, wherein adding comprisesintroducing the enzyme into the thin stillage, backset, or mixturethereof at a pH of about 3.0 to 9.0.
 38. The method of claim 26, whereinadding comprises introducing the agent into the thin stillage, backset,or mixture thereof at a temperature from about 30° C. to about 55° C.